Abstracts / Molecular Genetics and Metabolism 120 (2016) S17–S145
replacement therapy must be explored, considering the benefit of
the non-invasive nature of these evaluations.
doi:10.1016/j.ymgme.2016.11.049
41
Messenger RNA (mRNA) delivery to the liver corrects ornithine
transcarbamylase deficiency in a mouse disease model
Gordon Brandt, Mary Prieve, Eric Bell, Teri Blevins, Anna Galperin,
Pierrot Harvie, Allen Li, Jean-Rene Ella Menye, Sean Monahan, Amber
Paschal, Debashish Roy, Matt Waldheim, Michael Houston, PhaseRx,
Inc., Seattle, WA, United States
Introduction: Messenger RNA (mRNA) replacement is a promising
approach for treatment of single gene inherited metabolic diseases
such as ornithine transcarbamylase deficiency (OTCD). We report on
the development of a liver-targeted mRNA delivery platform which
has shown efficacy in multiple disease indications and the preclinical
proof of concept in a model of OTCD.
Methods: The definitive preclinical model for OTCD is the OTC spf ash
mouse model in which the mouse OTC mRNA is ablated resulting in
urea cycle dysfunction, rapid development of hyperammonemia, and
death. PRX-OTC, the liver-targeted mRNA drug product, replaces the
missing mouse OTC mRNA with the human OTC mRNA. PRX-OTC was
administered over 5 weeks at a dose of 3 mg/kg. Analyses included daily
body weights, urinary orotic acid, plasma ammonia, survival, and
human OTC protein expression in liver. Negative controls included
buffer and an mRNA drug product identical to PRX-OTC except for a
missing start codon.
Results: Animals treated with buffer or negative control rapidly
developed hyperammonemia and elevated urinary orotic acid, lost
body weight and had a median survival of 20 days. PRX-OTC
administration resulted in intra hepatic production of the human OTC
enzyme which persisted for more than 21 days after the final dose. PRX-
OTC treated animals showed normalization of plasma ammonia and
urinary orotic acid, gain of body weight, and complete survival through
the end of dosing (day 35) and for a further three weeks after dosing
(day 56). No elevations in cytokines were observed in groups receiving
PRX-OTC.
Conclusions: PRX-OTC administration corrects OTCD in the spf ash
model by replacing the human enzyme in the liver, thus normalizing
plasma ammonia and extending survival for weeks after dosing. Clinical
evaluation appears warranted.
doi:10.1016/j.ymgme.2016.11.050
42
Investigation of newborns screened in a pilot program for four
lysosomal diseases in Brazil
Heydy V Bravo-Villaltaa, Eurico C Netob, Jaqueline Schulteb, Jamile
Pereirab, Cláudio Sampaio-Filhoc, Maira G Burind, Fernanda H
Bitencourtd, Fernanda M Sebastiãod, Régis R Guidobonod, Ana C
Brusius-Facchind, Gabriela Pasqualima, Diana E Rojas-Málagaa, Ursula
S Mattea, Roberto Giuglianie, aUFRGS, Porto Alegre, - RS, Brazil, bCentro de
Triagem Neonatal, Porto Alegre, - RS, Brazil, cIntercientífica, São José dos
Campos, - SP, Brazil, dHospital de Clinicas de Porto Alegre, Porto Alegre, - RS,
Brazil, eHospital de Clinicas de Porto Alegre, Porto Alegre, Brazil
Newborn screening for lysosomal diseases has been gaining
considerable interest, as several of these conditions are now
S31
treatable and as early treatment seems to improve patient outcomes.
Also important, a number of different high-throughput platforms
are now available for this screening. We present the preliminary
results obtained with a fluorometric digital microfluidic platform to
perform multiplexed enzymatic analysis of α-L-iduronidase (IDUA,
to screen for MPS I), acid α-glucosidase (GAA, to screen for Pompe
disease), acid β-glucosidase (GBA, to screen for Gaucher disease) and
acid α-galactosidase (GLA, to screen for Fabry disease). The analyses
were performed in dried blood spot (DBS) samples from 10,527
newborns randomly selected among the cards routinely received by
the Neonatal Screening Center, based in Porto Alegre, Brazil. Time
from sample preparation to enzyme activity result on each cartridge
was less than 3 hours. Overall coefficient of variation (CV) values
between cartridges, days, instruments, and operators ranged from 4
to 20%; linearity correlation coefficients were ≥ 0.98 for all assays.
The method correctly discriminated samples of 9 affected patients
(3 MPS I, 2 Pompe, 2 Gaucher and 2 Fabry diseases) from samples
of normal babies. The analyses of all samples with activities below
cutoff were repeated. After retesting, 4 showed consistently low
enzyme activities (2 for IDUA, 1 for GLA and 1 for GBA). Further
investigation of these 4 cases, performed at Hospital de Clinicas,
Porto Alegre, Brazil, indicated 1 case of pseudodeficiency for MPS I,
1 case of pseudodeficiency for Pompe, 1 carrier for MPS I and 1
carrier for Gaucher. In conclusion, digital microfluidic technology
shows potential for the routine testing for the 4 lysosomal diseases
selected in a standard newborn screening laboratory. As in the
screening for other conditions, well-established algorithms should
be in place to quickly clarify any suspicion raised by the screening
results.
doi:10.1016/j.ymgme.2016.11.051
43
Sulfpraphane induces autophagy and reduces the level of mutated
huntingtin in human fibroblasts
Joanna Brokowska, Aleksandra Hac, Grzegorz Wegrzyn, Anna
Herman-Antosiewicz, University of Gdansk, Gdansk, Poland
Autophagy is a process of degradation of damaged organelles
and aggregates of proteins by lysosomal acid hydrolases, therefore,
lysosomes play a key role in this process. Autophagy can promotes
survival, whereas in other cases it appears to promote programmed
cell death. Disorders of autophagy may accompany many diseases
such as Huntington disease, Parkinson disease, Crohn disease, obesity
and mucopolysaccharidoses. It was demonstrated that in cancer cells
autophagy can be induced by sulforaphane (SFN), a natural agent
present in cruciferous plants. In our study, we determined whether
sulforaphane is able to induce autophagy in normal, non-cancerous
cells as well as to modulate the amount of mutated huntingtin in
human fibroblasts expressing the mutated protein. In cells treated
with sulforaphane, we observed reduction in the mutant huntingtin
level as well as a decrease in the amount of its aggregates comparing
to control cells. Simultaneously, we observed autophagy induction.
Our study revealed that SFN inhibited a major negative regulator
of autophagy, mTORC1. Autophagy induction and mTORC1 inhibi-
tion was preceded by activation of AMPK kinase, a known inhibitor
of mTORC1 and thus autophagy activator. The autophagy induction
by SFN coincided with a block in protein synthesis which might be,
together with the induction of autophagy, the molecular mecha-
nism leading to reduction of the mutant huntingtin amount in cells
by sulforaphane. Summarizing, our study shows that SFN induces
autophagy as well as inhibits protein synthesis and suggests that
sulforaphane can be used as potential therapeutic in diseases